Bruno L. Travi

Gender Is a Major Determinant of the Clinical Evolution and Immune Response in Hamsters Infected with Leishmania spp.

ABSTRACT In regions where leishmaniasis is endemic, clinical disease is usually reported more frequently among males than females. This difference could be due to disparate risks of exposure of males and females, but gender-related differences in the host response to infection may also play a role. Experimental studies of the influence of gender on Leishmania infection have not included parasites of the subgenus Viannia, which is the most common cause of cutaneous leishmaniasis in the Americas. Mice are not readily susceptible to infection by Leishmania (Viannia) spp., but cutaneous infection of hamsters with L. (V.) panamensis or L. (V.) guyanensis resulted in chronic lesions typical of the human disease caused by these parasites. Strikingly, infection of male hamsters resulted in significantly greater lesion size and severity, an increased rate of dissemination to distant cutaneous sites, and a greater parasite burden in the draining lymph node than infection in female animals. Two lines of evidence indicated this gender-related difference in disease evolution was determined at least in part by the sex hormone status of the animal. First, prepubertal male animals had smaller and/or less severe cutaneous lesions than adult male animals. Second, infection of testosterone-treated female animals resulted in significantly larger lesions than in untreated female animals. The increased severity of disease in male compared to female animals was associated with significantly greater intralesional expression of interleukin-4 (IL-4) (P = 0.04), IL-10 (P = 0.04), and transforming growth factor β (TGF-β) (P < 0.001), cytokines known to promote disease in experimental leishmaniasis. There was a direct correlation between the expression of TGF-β mRNA and lesion size (Spearmans correlation coefficient = 0.873; P < 0.001). These findings demonstrate an inherent risk of increased disease severity in male animals, which is associated with a more permissive immune response.

Spatial and Temporal Patterns of Phlebotomine Sand Flies (Diptera: Psychodidae) in a Cutaneous Leishmaniasis Focus in Northern Argentina

Abstract Phlebotomine sand flies (Diptera: Psychodidae) were captured in an area of Argentina endemic for American cutaneous leishmaniasis (ACL). A total of 44,944 flies were collected during a 130-wk interepidemic period from 1990 through 1993. These sand flies included Lutzomyia neivai (Pinto) (97.8%), Lutzomyia migonei (França) (1.2%), Lutzomyia cortelezzii (Brèthes) (0.8%), Lutzomyia shannoni (Dyar) (0.1%), and Lutzomyia punctigeniculata (Floch and Abonnenc) (0.1%). Lutzomyia neivai was more abundant in secondary forests and peridomestic environments associated with human cases than in primary forest or xeric thorn scrub areas. Time series analyses of species densities suggested a bimodal or trimodal annual pattern related to rainfall peaks, a 5-wk reproductive cycle, and peridomestic local populations that were located adjacent to secondary forests. In general, sand fly abundance was correlated with the rainfall of the previous year. Lutzomyia neivai spatial distributions were consistent with ACL incidence patterns during the study and in the recent outbreaks in Argentina. However, Lu. migonei also may be involved in peridomestic transmission. Our results suggest that there is a need for improved, long-term surveillance of sand flies and ACL cases, as well as development of effective intervention strategies.

Comparative vectorial efficiency of Lutzomyia evansi and Lu. longipalpis for transmitting Leishmania chagasi.

The infection rates and development of Leishmania chagasi in two sandfly species, Lutzomyia evansi and Lutzomyia longipalpis, were evaluated under natural and experimental conditions. Natural infection rates of Lu. evansi in San Andrés de Sotavento (Colombia) and Montañas de Peraza (Venezuela) (0.05 and 0.2%, respectively) were similar to those previously recorded for this species in Colombia and Venezuela and for Lu. longipalpis in many foci of American Visceral Leishmaniasis (AVL). Both sand fly species were able to support the development of two Colombian strains of L. chagasi experimentally acquired from dogs, hamsters or membrane feeders. However, the experimental infection rates and the sequence of parasite development in the guts of these sand flies revealed that parasite colonisation, differentiation, migration and attachment were more frequent and uniform in Lu. longipalpis than in Lu. evansi. This is consistent with a more recent association between L. chagasi and Lu. evansi, and these results might help to explain the irregularity of AVL outbreaks in foci where Lu. evansi has been reported as the sole vector.

Impact of habitat degradation on phlebotominae (Diptera: Psychodidae) of tropical dry forests in Northern Colombia.

Abstract We examined changes in the phlebotomine fauna resulting from human intervention in a tropical dry forest of Northern Colombia where visceral and cutaneous leishmaniases are endemic. A natural forest reserve (Colosó) and a highly degraded area (San Andrés de Sotavento [SAS]) were sampled monthly for 8 mo using Shannon traps, sticky traps, and resting-site collections. Overall abundances were higher in Colosó (15,988) than in SAS (2,324), and species richness of phlebotomines was greater in the forest reserve (11 species) than in the degraded habitat (seven species). Fisher alpha, a measure of diversity, reinforced this trend. Both sand fly communities were dominated by Lutzomyia evansi (Nuòez-Tovar), vector of Leishmania chagasi (Cunha & Chagas), representing 92 and 81% of all captures in Colosó and SAS, respectively. Lutzomyia longipalpis (Lutz & Neiva), the common vector of visceral leishmaniasis, accounted for 4–7% of the sand fly community. Lutzomyia panamensis (Shannon) and Lutzomyia gomezi (Nitzulescu), putative vectors of Leishmania braziliensis (Vianna), had low abundances at both study sites. The zoophilic species Lutzomyia cayennensis (Floch & Abonnenc) and Lutzomyia trinidadensis (Newstead) were present in variable numbers according to trapping methods and site. Habitat degradation negatively affected sand fly communities, but medically important species were able to exploit modified environments, thereby contributing to Leishmania endemicity.

Reduced Nitric Oxide Synthase 2 (NOS2) Promoter Activity in the Syrian Hamster Renders the Animal Functionally Deficient in NOS2 Activity and Unable to Control an Intracellular Pathogen

Progressive disease in the hamster model of visceral leishmaniasis, caused by Leishmania donovani, in contrast to infection in mice, mimics the progressive disease observed in untreated humans. During progressive infection in hamsters, there was a vigorous type 1 cellular immune response, which is typically associated with control of infection, suggesting that there was ineffective IFN-γ-mediated macrophage activation. Indeed, at the site of infection, hamsters did not express NO synthase 2 (NOS2), which is the primary mechanism for control of infection in mice. Furthermore, in striking contrast to mouse macrophages, IFN-γ-activated hamster macrophages did not did not express NOS2 nor generate NO, and were unable to restrict the replication of intracellular L. donovani. The absent hamster NOS2 expression was not the result of NOS2 gene deletion and the NOS2 cDNA had an intact open reading frame. Furthermore, the impaired transcription of NOS2 mRNA was selective and not due to global impairment of IFN-γ signaling (members of the IFN-γ-signaling pathway were expressed and functional and IFN-γ up-regulated several primary and secondary response genes). Strikingly, the proximal hamster NOS2 promoter, like the human ortholog, had >20-fold less basal and IFN-γ/LPS-inducible activity than the corresponding mouse promoter. Thus, reduced basal and IFN-γ-induced activity of the hamster NOS2 transcriptional unit, which is unique to this small animal and similar to the human counterpart, accompanies the inability of the animal to control an intracellular pathogen.

Dynamics of Leishmania chagasi infection in small mammals of the undisturbed and degraded tropical dry forests of northern Colombia

The infection rate with Leishmania chagasi and the population dynamics of small mammals were studied in an undisturbed forest reserve (Colosó) and an area of highly degraded forest (San Andrés de Sotavento [SAS]) in northern Colombia, both endemic for visceral leishmaniasis. Live trapping of mammals was done every month, and species, age, sex and reproductive status determined. L. chagasi was detected in samples of skin or spleen by the polymerase chain reaction, after extraction of deoxyribonucleic acid using specific primers (DB8/AJS3), and dot blood hybridization. Didelphis marsupialis was found to be infected in Colosó (3/21, 14.3%) and SAS (13/137, 9.5%); its relative abundance was higher in SAS (93/113, 82% of the captures). Although Proechimys canicollis was also found to be infected in Colosó (3/34, 8.8%) and SAS (2/4), its relative abundance was much lower (4%) in SAS than in Colosó (56% of 77 animals captured). Sciurus granatensis, Marmosa robinsoni, Heteromys anomalus, Zygodontomys brevicauda and Metachirus nudicaudatus were less common, and no L. chagasi infection was detected in them.

Metastatic capability of Leishmania (viannia) panamensis and Leishmania (viannia) guyanensis in golden hamsters

The pattern and kinetics of internal dissemination and frequency of cutaneous metastatic lesions resulting from experimental infection of golden hamsters with Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis were examined. Nineteen strains were evaluated: 16 L. (V.) panamensis isolated from patients and 3 L. (V.) guyanensis, 2 isolated from human cases and 1 WHO reference strain originating from a sandfly vector. Lymphatic dissemination occurred within 3 mo and was observed for 16 of 16 (100%) of L. (V.) panamensis and 3 of 3 (100%) of L. (V.) guyanensis. Parasites were cultured infrequently from liver and spleen: 3 of 125 (2%) L. (V.) panamensis and 1 of 22 (5%) L. (V.) guyanensis. Decreased frequency of isolation from the inoculation site and draining lymph nodes over time was accompanied by increased frequency of isolation from distant lymph nodes. Dilution of triturated tissue samples resulted in an increased efficiency of parasite culture. Both primary lesions and secondary cutaneous metastatic lesions were more severe in hamsters infected with L. (V.) guyanensis than with L. (V.) panamensis. Cutaneous metastatic lesions were produced more frequently by L. (V.) guyanensis, 24 of 46 hamsters (52%), than by L. (V.) panamensis, 28 of 252 hamsters (11%). Individual Leishmania strains displayed distinctive propensities to produce cutaneous metastases, manifested as a reproducible phenotype. Metastatic pathogenicity was independent of the inoculum dose, supporting the dissociation of infectivity and pathogenicity.

Pregnancy enhances the innate immune response in experimental cutaneous leishmaniasis through hormone-modulated nitric oxide production

The maintenance of host defense during pregnancy may depend on heightened innate immunity. We evaluated the immune response of pregnant hamsters during early infection with Leishmania (Viannia) panamensis, a cause of American cutaneous leishmaniasis. At 7 days post‐infection, pregnant animals showed a lower parasite burden compared with nonpregnant controls at the cutaneous infection site (P=0.0098) and draining lymph node (P=0.02). Resident peritoneal macrophages and neutrophils from pregnant animals had enhanced Leishmania killing capacity compared with nonpregnant controls (P=0.018 each). This enhanced resistance during pregnancy was associated with increased expression of inducible NO synthase (iNOS) mRNA in lymph node cells (P=0.02) and higher NO production by neutrophils (P=0.0001). Macrophages from nonpregnant hamsters infected with L. panamensis released high amounts of NO upon estrogen exposure (P=0.05), and addition of the iNOS inhibitor L‐N6‐(1‐iminoethyl) lysine blocked the induction of NO production (P=0.02). Infected, nonpregnant females treated with estrogen showed a higher percentage of cells producing NO at the infection site than controls (P=0.001), which correlated with lower parasite burdens (P=0.036). Cultured macrophages or neutrophils from estrogen‐treated hamsters showed significantly increased NO production and Leishmania killing compared with untreated controls. iNOS was identified as the likely source of estrogen‐induced NO in primed and naïve macrophages, as increased transcription was evident by real‐time PCR. Thus, the innate defense against Leishmania infection is heightened during pregnancy, at least in part as a result of estrogen‐mediated up‐regulation of iNOS expression and NO production.

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection.

Clinical, Parasitologic, and Immunologic Evolution in Dogs Experimentally Infected with Sand Fly-Derived Leishmania chagasi Promastigotes

Experimental infection of dogs with Leishmania infantum has yielded heterogeneous clinical, parasitologic, and immunologic results. We studied dogs infected with 10(5) or 10(4) sand fly-derived promastigotes delivered by the intradermal (ID) or intravenous (IV) routes. Total mortality over 1 year post-infection reached 23.8%. The mortality and proportion of sustained polysymptomatic dogs was highest in the IV-10(5) group. The early appearance of polysymptoms was associated with an increased risk of progression to death. Dissemination of the parasite to lymph nodes was faster, and the subsequent infectivity to sand flies higher, in the IV compared with ID-infected dogs. Parasite-specific IgG1 or IgG2 production was similar among the groups, but higher interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) expression was associated with polysymptomatic dogs. On the basis of the data obtained from this study, a sample size analysis using different endpoints for future vaccine trials is described.